Long-term Dynamics of Fungal Communities Inhabiting Decaying Stumps of Quercus robur.

We studied the diversity, composition, and long-term dynamics of wood-inhabiting fungi in Quercus robur stumps left after commercial tree harvesting in Lithuania. Sampling of wood was carried out at three sites and from stumps, which were 10-, 20-, 30-, 40-, and 50-year-old. DNA was isolated from wood samples and fungal communities analyzed using high-throughput sequencing. Results showed that stump age had a limited effect on fungal diversity. The development of fungal communities in oak stums was found to be a slow process as fungal communities remained similar for decades, while larger changes were only detected in older stumps. The most common fungi were Eupezizella sp. (18.4%), Hyphodontia pallidula (12.9%), Mycena galericulata (8.3%), and Lenzites betulinus (7.1%). Fistulina hepatica, which is a red-listed wood-decay oak fungus, was also detected at a low relative abundance in stump wood. In the shortage of suitable substrate, oak stumps may provide habitats for long-term survival of different fungal species, including red-listed and oak-related fungi.

In vitro evidence of root colonization suggests ecological versatility in the genus Mycena.

The root-associated habit has evolved on numerous occasions in different fungal lineages, suggesting a strong evolutionary pressure for saprotrophic fungi to switch to symbiotic associations with plants. Species within the ubiquitous, saprotrophic genus Mycena are frequently major components in molecular studies of root-associated fungal communities, suggesting that an evaluation of their trophic status is warranted. Here, we report on interactions between a range of Mycena species and the plant Betula pendula. In all, 17 Mycena species were inoculated onto B. pendula seedlings. Physical interactions between hyphae and fine roots were examined using differential staining and fluorescence microscopy. Physiological interactions were investigated using 14 C and 32 P to show potential transfer between symbionts. All Mycena species associated closely with fine roots, showing hyphal penetration into the roots, which in some cases were intracellular. Seven species formed mantle-like structures around root tips, but none formed a Hartig net. Mycena pura and Mycena galopus both enhanced seedling growth, with M. pura showing significant transfer of 32 P to the seedlings. Our results support the view that several Mycena species can associate closely with plant roots and some may potentially occupy a transitional state between saprotrophy and biotrophy.

Secondary Metabolites from the Root Rot Biocontrol Fungus Phlebiopsis gigantea.

Three cyclopentanoids (phlebiopsin A⁻C), one glycosylated p-terphenyl (methyl-terfestatin A), and o-orsellinaldehyde were isolated from the biocontrol fungus Phlebiopsis gigantea, and their structures were elucidated by 1D and 2D NMR spectroscopic analysis, as well as by LC-HRMS. The biological activity of the compounds against the root rot fungus Heterobasidion occidentale, as well as against Fusarium oxysporum and Penicillium canescens, was also investigated, but only o-orsellinaldehyde was found to have any antifungal activity in the concentration range tested.

Growing evidence for facultative biotrophy in saprotrophic fungi: data from microcosm tests with 201 species of wood-decay basidiomycetes.

Ectomycorrhizal (ECM) symbioses have evolved a minimum of 78 times independently from saprotrophic lineages, indicating the potential for functional overlap between ECM and saprotrophic fungi. ECM fungi have the capacity to decompose organic matter, and although there is increasing evidence that some saprotrophic fungi exhibit the capacity to enter into facultative biotrophic relationships with plant roots without causing disease symptoms, this subject is still not well studied. In order to determine the extent of biotrophic capacity in saprotrophic wood-decay fungi and which systems may be useful models, we investigated the colonization of conifer seedling roots in vitro using an array of 201 basidiomycete wood-decay fungi. Microtome sectioning, differential staining and fluorescence microscopy were used to visualize patterns of root colonization in microcosm systems containing Picea abies or Pinus sylvestris seedlings and each saprotrophic fungus. Thirty-four (16.9%) of the tested fungal species colonized the roots of at least one tree species. Two fungal species showed formation of a mantle and one showed Hartig net-like structures. These features suggest the possibility of an active functional symbiosis between fungus and plant. The data indicate that the capacity for facultative biotrophic relationships in free-living saprotrophic basidiomycetes may be greater than previously supposed.

Impact of Heterobasidion root-rot on fine root morphology and associated fungi in Picea abies stands on peat soils.

We examined differences in fine root morphology, mycorrhizal colonisation and root-inhabiting fungal communities between Picea abies individuals infected by Heterobasidion root-rot compared with healthy individuals in four stands on peat soils in Latvia. We hypothesised that decreased tree vitality and alteration in supply of photosynthates belowground due to root-rot infection might lead to changes in fungal communities of tree roots. Plots were established in places where trees were infected and in places where they were healthy. Within each stand, five replicate soil cores with roots were taken to 20 cm depth in each root-rot infected and uninfected plot. Root morphological parameters, mycorrhizal colonisation and associated fungal communities, and soil chemical properties were analysed. In three stands root morphological parameters and in all stands root mycorrhizal colonisation were similar between root-rot infected and uninfected plots. In one stand, there were significant differences in root morphological parameters between root-rot infected versus uninfected plots, but these were likely due to significant differences in soil chemical properties between the plots. Sequencing of the internal transcribed spacer of fungal nuclear rDNA from ectomycorrhizal (ECM) root morphotypes of P. abies revealed the presence of 42 fungal species, among which ECM basidiomycetes Tylospora asterophora (24.6 % of fine roots examined), Amphinema byssoides (14.5 %) and Russula sapinea (9.7 %) were most common. Within each stand, the richness of fungal species and the composition of fungal communities in root-rot infected versus uninfected plots were similar. In conclusion, Heterobasidion root-rot had little or no effect on fine root morphology, mycorrhizal colonisation and composition of fungal communities in fine roots of P. abies growing on peat soils.

The ectomycorrhizal community of conifer stands on peat soils 12 years after fertilization with wood ash.

We studied long-term effects of fertilization with wood ash on biomass, vitality and mycorrhizal colonization of fine roots in three conifer forest stands growing in Vacciniosa turf. mel. (V), Myrtillosa turf. mel. (M) and Myrtillosa turf. mel./Caricoso-phragmitosa (MC) forest types on peat soils. Fertilization trials amounting 5 kg/m(2) of wood ash were established 12 years prior to this study. A total of 63 soil samples with roots were collected and analysed. Ectomycorrhizal (ECM) fungi in roots were identified by morphotyping and sequencing of the fungal internal transcribed spacer (ITS) region. In all forest types, fine root biomass was higher in fertilized plots than in control plots. In M forest type, proportion of living fine roots was greater in fertilized plots than in control plots, while in V and MC, the result was opposite. Fifty ECM species were identified, of which eight were common to both fertilized and control plots. Species richness and Shannon diversity index were generally higher in fertilized plots than in control plots. The most common species in fertilized plots were Amphinema byssoides (17.8%) and Tuber cf. anniae (12.2%), while in control plots, it was Tylospora asterophora (18.5%) and Lactarius tabidus (20.3%). Our results showed that forest fertilization with wood ash has long-lasting effect on diversity and composition of ECM fungal communities.

High-Throughput Sequencing Reveals Drastic Changes in Fungal Communities in the Phyllosphere of Norway Spruce (Picea abies) Following Invasion of the Spruce Bud Scale (Physokermes piceae).

The aim of this study was to assess the diversity and composition of fungal communities in damaged and undamaged shoots of Norway spruce (Picea abies) following recent invasion of the spruce bud scale (Physokermes piceae) in Lithuania. Sampling was done in July 2013 and included 50 random lateral shoots from ten random trees in each of five visually undamaged and five damaged 40-50-year-old pure stands of P. abies. DNA was isolated from 500 individual shoots, subjected to amplification of the internal transcribed spacer of fungal ribosomal DNA (ITS rDNA), barcoded and sequenced. Clustering of 149,426 high-quality sequences resulted in 1193 non-singleton contigs of which 1039 (87.1 %) were fungal. In total, there were 893 fungal taxa in damaged shoots and 608 taxa in undamaged shoots (p < 0.0001). Furthermore, 431 (41.5 %) fungal taxa were exclusively in damaged shoots, 146 (14.0 %) were exclusively in undamaged shoots, and 462 (44.5 %) were common to both types of samples. Correspondence analysis showed that study sites representing damaged and undamaged shoots were separated from each other, indicating that in these fungal communities, these were largely different and, therefore, heavily affected by P. piceae. In conclusion, the results demonstrated that invasive alien tree pests may have a profound effect on fungal mycobiota associated with the phyllosphere of P. abies, and therefore, in addition to their direct negative effect owing physical damage of the tissue, they may also indirectly determine health, sustainability and, ultimately, distribution of the forest tree species.

Cytotoxic Illudane Sesquiterpenes from the Fungus Granulobasidium vellereum (Ellis and Cragin) Jülich.

Eight illudane sesquiterpenes were obtained from the wood-decomposing fungus Granulobasidium vellereum (Ellis and Cragin) Jülich; among them were the enantiomers of the known compounds illudin M (1) and dihydroilludin M (4) and the diastereomers of illudin M (2) and illudin S (3), as well as two previously undescribed illudanes (5, 6). The cytotoxicity of compounds 1-4 and 6 was evaluated against two tumor cell lines (Huh7 and MT4), which showed that compounds 1-3 had potent cytotoxic activity, whereas compounds 4 and 6 had no or only moderate effects at concentrations up to 400 µM. Surprisingly, both compounds 2 and 3 were about 10 times more potent than 1. When the chemical reactivity of 1 and 2 was tested, compound 2 was shown to have a substantially higher reaction rate when reacted both with 2 M HCl and with cysteine, indicating that the difference in cytotoxicity is probably due to chemical reactivity and not to enzymatic affinity.

Large-scale introgression shapes the evolution of the mating-type chromosomes of the filamentous ascomycete Neurospora tetrasperma.

The significance of introgression as an evolutionary force shaping natural populations is well established, especially in animal and plant systems. However, the abundance and size of introgression tracts, and to what degree interspecific gene flow is the result of adaptive processes, are largely unknown. In this study, we present medium coverage genomic data from species of the filamentous ascomycete Neurospora, and we use comparative genomics to investigate the introgression landscape at the genomic level in this model genus. We revealed one large introgression tract in each of the three investigated phylogenetic lineages of Neurospora tetrasperma (sizes of 5.6 Mbp, 5.2 Mbp, and 4.1 Mbp, respectively). The tract is located on the chromosome containing the locus conferring sexual identity, the mating-type (mat) chromosome. The region of introgression is confined to the region of suppressed recombination and is found on one of the two mat chromosomes (mat a). We used Bayesian concordance analyses to exclude incomplete lineage sorting as the cause for the observed pattern, and multilocus genealogies from additional species of Neurospora show that the introgression likely originates from two closely related, freely recombining, heterothallic species (N. hispaniola and N. crassa/N. perkinsii). Finally, we investigated patterns of molecular evolution of the mat chromosome in Neurospora, and we show that introgression is correlated with reduced level of molecular degeneration, consistent with a shorter time of recombination suppression. The chromosome specific (mat) and allele specific (mat a) introgression reported herein comprise the largest introgression tracts reported to date from natural populations. Furthermore, our data contradicts theoretical predictions that introgression should be less likely on sex-determining chromosomes. Taken together, the data presented herein advance our general understanding of introgression as a force shaping eukaryotic genomes.

Root-associated fungi of Rosa rugosa grown on the frontal dunes of the Baltic Sea Coast in Lithuania.

The aim of the present study was to assess fungal communities associated with fine living roots of Rosa rugosa Thunb grown on the frontal dunes of Curonian Spit at the Baltic Sea coast in Lithuania. The roots of R. rugosa were sampled at five sites (Nida, Preila, Pervalka, Juodkrante and Smiltyne) situated at a distance ca. 5-15 km from each other. Direct amplification, cloning and sequencing of fungal ITS rRNA from the fine roots resulted in 134 high-quality sequences, representing 31 fungal taxa among which saprotrophs and endophytes Mycena sp. (14.2 %), Tumularia sp. (14.2 %), Penicillium spinulosum (11.9 %) and Cadophora malorum (9.0 %) were most common. Arbuscular mycorrhizal fungi including Entrophospora baltica (0.7 %) and Rhizophagus irregularis (0.7 %) and potentially root pathogenic fungi--Ceratobasidium sp. (4.5 %), Fusarium oxysporum (3.0 %), Fusarium culmorum (0.7 %) and Ilyonectria crassa (0.7 %)--were also detected at low proportions. In conclusion, the results demonstrated that the fine roots of R. rugosa are inhabited by various groups of fungi. Although saprotrophs and endophytes were dominant, the detection of arbuscular mycorrhizal fungi indicated that these may be important for mineral nutrition of R. rugosa established on dry and poor fertility coastal dunes.

Cytotoxic illudalane sesquiterpenes from the wood-decay fungus Granulobasidium vellereum (Ellis & Cragin) Jülich.

Seven illudalane sesquiterpenes were obtained from the wood decomposing fungus Granulobasidium vellereum: granuloinden A, granuloinden B and dihydrogranuloinden, along with the previously known compounds radulactone, pterosin M, echinolactone A and D. Granuloinden B showed potent cytotoxic activity against the Huh7 and MT4 tumor cell lines (CC50 values of 6.7 and 0.15 µM, respectively), whereas granuloinden A and dihydrogranuloinden had no or moderate activity.

Fungal Communities Associated with Siricid Wood Wasps: Focus on Sirex juvencus, Urocerus gigas, and Tremex fuscicornis.

We investigated the diversity and occurrence of wood wasps in Lithuania and determined communities of associated fungi. Trapping of wood wasps resulted in three different species, including Sirex juvencus, Urocerus gigas, and Tremex fuscicornis. Fungal culturing from adult females of T. fuscicornis mainly resulted in fungi from the genera Penicillium and Trichoderma. High-throughput sequencing of ITS2 rDNA resulted in 59,797 high-quality fungal sequences, representing 127 fungal OTUs. There were 93 fungal OTUs detected in U. gigas, 66 in S. juvencus, and 10 in T. fuscicornis. The most common fungi were Fusarium sporotrichioides (63.1% of all fungal sequences), Amylostereum chailletii (14.9%), Penicillium crustosum (7.8%), Microascus sp. 2261_4 (5.0%), and Pithoascus ater (2.1%). Among these, only A. chailletii was found in all three insect species with the highest relative abundance in U. gigas (15.2%), followed by S. juvencus (7.7%), and the lowest in T. fuscicornis (0.3%) (p < 0.0003). Correspondence analysis of fungal communities showed a distant placement of different species of wood wasps, indicating that fungal communities in each of these were largely different. In conclusion, the study showed that the economically important tree pathogen A. chailletii was among the most common fungal OTUs vectored by siricid wood wasps.

An Overview of Phytophthora Species on Woody Plants in Sweden and Other Nordic Countries.

The genus Phytophthora, with 326 species in 12 phylogenetic clades currently known, includes many economically important pathogens of woody plants. Different Phytophthora species often possess a hemibiotrophic or necrotrophic lifestyle, have either a broad or narrow host range, can cause a variety of disease symptoms (root rot, damping-off, bleeding stem cankers, or blight of foliage), and occur in different growing environments (nurseries, urban and agricultural areas, or forests). Here, we summarize the available knowledge on the occurrence, host range, symptoms of damage, and aggressiveness of different Phytophthora species associated with woody plants in Nordic countries with a special emphasis on Sweden. We evaluate the potential risks of Phytophthora species to different woody plants in this geographical area and emphasize the increasing threats associated with continued introduction of invasive Phytophthora species.

Biogeography of Fungal Communities Associated with Pinus sylvestris L. and Picea abies (L.) H. Karst. along the Latitudinal Gradient in Europe.

We assessed the diversity and composition of fungal communities in different functional tissues and the rhizosphere soil of Pinus sylvestris and Picea abies stands along the latitudinal gradient of these tree species distributions in Europe to model possible changes in fungal communities imposed by climate change. For each tree species, living needles, shoots, roots, and the rhizosphere soil were sampled and subjected to high-throughput sequencing. Results showed that the latitude and the host tree species had a limited effect on the diversity and composition of fungal communities, which were largely explained by the environmental variables of each site and the substrate they colonize. The mean annual temperature and mean annual precipitation had a strong effect on root fungal communities, isothermality on needle fungal communities, mean temperature of the warmest quarter and precipitation of the driest month on shoot fungal communities, and precipitation seasonality on soil fungal communities. Fungal communities of both tree species are predicted to shift to habitats with a lower annual temperature amplitude and with increasing precipitation during the driest month, but the suitability of these habitats as compared to the present conditions is predicted to decrease in the future.

Fungal Communities in Leaves and Roots of Healthy-Looking and Diseased Ulmusglabra.

The aim of this study was to investigate fungal communities associated with leaves and roots of healthy-looking and declining U. glabra trees. The study was expected to demonstrate whether and how the diversity and composition of fungal communities change in these functional tissues following the infection by Dutch elm disease-causing fungi. The study sites included six U. glabra sites in Lithuania, where leaves and roots were sampled. DNA was isolated from individual samples, amplified using ITS2 rRNA as a marker, and subjected to high-throughput sequencing. The sequence analysis showed the presence of 32,699 high-quality reads, which following clustering, were found to represent 520 non-singleton fungal taxa. In leaves, the fungal species richness was significantly higher in healthy-looking trees than in diseased ones (p < 0.05). In roots, a similar comparison showed that the difference was insignificant (p > 0.05). The most common fungi in all samples of roots were Trichocladium griseum (32.9%), Penicillium restrictum (21.2%), and Unidentified sp. 5238_7 (12.6%). The most common fungi in all samples of leaves were Trichomerium sp. 5238_8 (12.30%), Aureobasidium pullulans (12.03%), Cladosporium sp. 5238_5 (11.73%), and Vishniacozyma carnescens (9.86%). The results showed that the detected richness of fungal taxa was higher in samples collected from healthy-looking trees than from diseased ones, thereby highlighting the negative impact of the Dutch elm disease on the overall fungal diversity.

Principal Drivers of Fungal Communities Associated with Needles, Shoots, Roots and Adjacent Soil of Pinus sylvestris.

The plant- and soil-associated microbial communities are critical to plant health and their resilience to stressors, such as drought, pathogens, and pest outbreaks. A better understanding of the structure of microbial communities and how they are affected by different environmental factors is needed to predict and manage ecosystem responses to climate change. In this study, we carried out a country-wide analysis of fungal communities associated with Pinus sylvestris growing under different environmental conditions. Needle, shoot, root, mineral, and organic soil samples were collected at 30 sites. By interconnecting the high-throughput sequencing data, environmental variables, and soil chemical properties, we were able to identify key factors that drive the diversity and composition of fungal communities associated with P. sylvestris. The fungal species richness and community composition were also found to be highly dependent on the site and the substrate they colonize. The results demonstrated that different functional tissues and the rhizosphere soil of P. sylvestris are associated with diverse fungal communities, which are driven by a combination of climatic (temperature and precipitation) and edaphic factors (soil pH), and stand characteristics.

Fungal Communities in Re-Emerging Fraxinus excelsior Sites in Lithuania and Their Antagonistic Potential against Hymenoscyphus fraxineus.

Fifty-nine fungal taxa, isolated from re-emerging Fraxinus excelsior sites in Lithuania, were in vitro tested against three strains of Hymenoscyphus fraxineus on agar media to establish their biocontrol properties. All tested fungi were isolated from leaves and shoots of relatively healthy Fraxinus excelsior trees (<30% defoliation), which were affected by ash dieback but their phytosanitary condition has not worsened during the last decade. The inhibition of H. fraxineus growth by tested fungal taxa ranged between 16−87%. Occasionally isolated fungal taxa such as Neonectria coccinea, Nothophorma quercina, and Phaeosphaeria caricis were among the most effective fungi inhibiting the growth of H. fraxineus cultures. Among the more commonly isolated fungal taxa, Cladosporium sp., Fusarium sp., Malassezia sp., and Aureobasidium pullulans showed a strong growth inhibition of H. fraxineus.

Fungi in roots of nursery grown Pinus sylvestris: ectomycorrhizal colonisation, genetic diversity and spatial distribution.

The aims of this study were to investigate patterns of ectomycorrhizal (ECM) colonisation and community structure on nursery grown seedlings of Pinus sylvestris, spatial distribution of ECMs in the nursery plot and genetic diversity of commonly isolated ECM basidiomycete Hebeloma cavipes. One hundred seedlings were sampled in 225 m(2) area using a systematic grid design. For each seedling, 20 individual root tips were randomly collected, morphotyped, and surface sterilised for fungal isolation in pure culture. Results showed that ECM community was comprised of nine distinct morphotypes among which Thelephora terrestris (39.7%), Hebeloma sp. (17.8%) and Suillus luteus (6.1%) were the most abundant. Spatial distribution of ECMs in the nursery plot was determined by their relative abundance: even in common ECMs and random in rare ones. Fungal isolation yielded 606 pure cultures, representing 71 distinct taxa. The most commonly isolated fungi were the ascomycetes Neonectria macrodidyma (20.3%), Phialocephala fortinii (13.5%), Neonectria radicicola (6.3%) and the ECM basidiomycete H. cavipes (4.5%). Intraspecific genetic diversity within 27 H. cavipes isolates was studied using two methods: restriction digestion of the amplified intergenic spacer of nuclear ribosomal DNA and genealogical concordance of five genetic markers. Five and eight genotypes were revealed by each respective method, but both of those were largely consistent, in particular, in determining the largest genotype (A) composed of 18 isolates. Mapping positions for each H. cavipes isolate and genotype in the field showed that isolates of the A genotype covered a large part of the nursery plot. This suggests that H. cavipes is largely disseminated by vegetative means of local genotypes and that nursery cultivation practices are likely to contribute to the dissemination of this species in the forest nursery soils.

The mating-type chromosome in the filamentous ascomycete Neurospora tetrasperma represents a model for early evolution of sex chromosomes.

We combined gene divergence data, classical genetics, and phylogenetics to study the evolution of the mating-type chromosome in the filamentous ascomycete Neurospora tetrasperma. In this species, a large non-recombining region of the mating-type chromosome is associated with a unique fungal life cycle where self-fertility is enforced by maintenance of a constant state of heterokaryosis. Sequence divergence between alleles of 35 genes from the two single mating-type component strains (i.e. the homokaryotic mat A or mat a-strains), derived from one N. tetrasperma heterokaryon (mat A+mat a), was analyzed. By this approach we were able to identify the boundaries and size of the non-recombining region, and reveal insight into the history of recombination cessation. The non-recombining region covers almost 7 Mbp, over 75% of the chromosome, and we hypothesize that the evolution of the mating-type chromosome in this lineage involved two successive events. The first event was contemporaneous with the split of N. tetrasperma from a common ancestor with its outcrossing relative N. crassa and suppressed recombination over at least 6.6 Mbp, and the second was confined to a smaller region in which recombination ceased more recently. In spite of the early origin of the first "evolutionary stratum", genealogies of five genes from strains belonging to an additional N. tetrasperma lineage indicate independent initiations of suppressed recombination in different phylogenetic lineages. This study highlights the shared features between the sex chromosomes found in the animal and plant kingdoms and the fungal mating-type chromosome, despite fungi having no separate sexes. As is often found in sex chromosomes of plants and animals, recombination suppression of the mating-type chromosome of N. tetrasperma involved more than one evolutionary event, covers the majority of the mating-type chromosome and is flanked by distal regions with obligate crossovers.

First Report of the Larch Longhorn (Tetropium gabrieli Weise, Coleoptera: Cerambycidae: Spondylidinae) on Larix spp. in Lithuania.

Tetropium gabrieli is known to be native to the Alps in Europe where it breeds in European larch (Larix decidua), but it has spread to other areas and was reported in Poland, Sweden, Denmark and Belorussia. Although T. gabrieli is considered an important secondary pest of Larix spp., it can be particularly harmful to trees subjected to abiotic stress. Here we report that in Lithuania, T. gabrieli was for the first time captured in 2019 using sticky traps attached to Larix spp. trees. Two adult beetles were trapped at two different sites in central Lithuania, and this was in the period between 10th of May and 5th of June. Regarding potential threats caused by this insect pest, this new finding requires special attention, particularly on its biology, ecology, and local distribution.